Behaviour of mouse macrophage cell line and mouse fibroblast on copolymers containing cellulose triacetate

Behaviour of mouse macrophage cell line and mouse fibroblast on copolymers containing cellulose triacetate Yoshihiro Shigemasa, Hitoshi Sashiwa, Shin-ichiro Tanioka and Hiroyuki Saimito Department of Materials Science, Faculty of Engineerin 9, Tot tori University, Koyama-cho, Tottori 680, Japan

Takahiko Tanigawa and Yoshinori Tanaka Department of Bacteriology, Faculty of Medicine, Tottori University, Yonago 683, Japan

(Received 22 November 1991; revised 17 April 1992) Copolymers containing cellulose triaeetate were prepared under various conditions. A bacteriological plastic dish was coated with the copolymer ( HCTA-MDI copolymer) composed of hydrolysed cellulose triacetate (HCTA) and diphenylmethane diisocyanate (MDI). The deacetylated copolymer (DA-copolymer) was prepared by the deaeetylation of HCTA-MDI copolymer. The behaviour of animal cells such as a mouse macrophage cell line ( A640-BB-2 cells) and a mouse fibroblast (3T6 cells) on prepared dishes was investigated morphologically. A640-BB-2 cells showed good adhesion and smooth spreading, and 3T6 cells also showed good adhesion and sufficient cell growth on the dish coated with HCTA-MDI copolymer. These results suggest that these copolymers are useful for biomedical materials. Keywords: Copolymer;cellulosetriacetate;diphenylmethanediisocyanate;mousemacrophage;mousefibroblast

Introduction Block and graft copolymers composed of polysaccharide have become of considerable interest because of their many unique properties. Steinmann 1, Kim 2, and Amick 3 have prepared block copolymers by coupling various diisocyanates with cellulose triacetate oligomeric species having hydroxyl end-groups. These copolymers are expected to show novel properties such as elastometric ~, biodegradable 2, and water sorption properties 3. Our interest is in the application of copolymers composed of various polysaccharides as biomedical materials. However, polyurethanes are well known to be anticoagulant materials and widely used as biomedical materials4. In general, these polyurethanes are prepared from diisocyanates and diols such as poly(ethylene glycol), poly(propylene glycol), and so on 5. However, it has been pointed out that these polyurethanes are toxic to animals6. Recently, the preparation of some biomedical polymers containing saccharide has been investigated. Kobayashi reported that lactose-substituted styrene homopolymer (PVLA) was useful as a culture substratum of hepatocytes7. Moreover, it has been clarified that carbohydrate plays an important role in the recognition of mutual interaction of cells s. Furthermore, biomaterials must be safe in vivo and that means biocompatible. Compatibility with animal cells is required for the biomedical use of these polymers6. Safety of a material for animals is usually evaluated by the multiplication and attachment of cells to the material in tissue culture. Taking these points into consideration, the 0141-8130/92/050274-05 © 1992Butterworth-HeinemannLimited 274 Int. J. Biol. Macromol., 1992, Vol. 14, October

polymers containing carbohydrate are expected to be biocompatible. In this study, we prepared copolymers containing cellulose triacetate (HCTA-MDI copolymer: Scheme 1 ) and observed morphologically the behaviour of animal cells such as a mouse macrophage cell line (A640-BB-2 cells) and a mouse fibroblast (3T6 cells) on the dish coated with HCTA-MDI copolymer.

Experimental Materials

Cellulose triacetate (d.s., 2.9; MW, 86 000), Dulbecco's modifed Eagle's medium (DEM) with 0.1% D-glucose, and other reagents were purchased from Daicel Chemical Industries Ltd, Grand Island Biological Co., USA and Wako Pure Chemical Industries Ltd, respectively. These reagents were used without further purification. Hydrolys& o f cellulose triacetate

Cellulose triacetate was hydrolysed according to the method of Steinmann ~ as follows: cellulose triacetate (20 g) was dissolved in 197 ml of acetic acid at room temperature. Acetic anhydride (3.3 ml), c o n c . H 2 S O 4 (1.0 ml), and water (1.2 ml ) were added to the solution in this order and the mixture was heated to 83°C for 7 h. After cooling to room temperature, the mixture was poured into 1200ml of water. The precipitate was collected by centrifugation, washed with water, and dried

Copolymers containing cellulose triacetate: Y. Shigemasa et al.

OCNR'NCO _ L '-

-'1"-

"OR~

L ~r " r~ - . s = . .CH2OR ~'-'. ^ "TI ~ _ \

[L R O " ~ ' ~ ~ O R

jn

0,, /] /" 0. L.,..-CNR'NCO-I--05n H H Jm

R= -CCH 3 0 HCTA

HCTA-MDI

copolymer

Scheme 1

in vacuo at room temperature to give 12 g of white powder (hydrolysed cellulose triacetate). Copolymerization of hydrolysed cellulose triacetate and diisocyanate A typical procedure of copolymerization is as follows: hydrolysed cellulose triacetate (HTAC; 1.0g) was dissolved in CHEC12 (20 ml) in a 30-ml flask fitted with an agitator and a condenser under a nitrogen atmosphere. The solution was heated with agitation and about 10 ml of the solvent was azeotropically distilled to dry the system. Diphenylmethane diisocyanate (MDI, 0.5mmol) and triethylamine (0.05mmol) in CHzC12 were added to the solution stepwisely at various time intervals. After stirring at 33°C under a nitrogen atmosphere for 48 h, the reaction was quenched by the addition of N,N-dimethylformamide (DMF, 8 ml) and water (1 ml), followed by pouring the reaction mixture into ether (200 ml). The precipitate formed was collected by centrifugation, washed with acetone, and dried in 1)acuo.

Yield: 70% =

HCTA-MDI copolymer (g) × 100 HCTA added (g) + MDI added (g)

Molecular weight determination Molecular weights of HCTA and HCTA-MDI copolymers were determined by g.p.c, with pullulane as the standard (column, G2000HxL, G2500HxL, and G5000HxL TSK gel-columns; eluent 5 mM LiBr in DMF; flow rate 1.0 ml/min; column temp, 49°C; detector, RI). Preparation of dish coated with copolymer The copolymer prepared was dissolved in dimethylsulphoxide (20 mg/ml) and the solution was poured into a tissue culture plastic dish (35 × 10mm, Falcon Labware, Div. Becton Dickinson Oversea, Oxnard, CA, USA; Catalogue No. 3001). The solution was dried in vacuo at 50°C and the dish was washed with water to remove the solvent efficiently. The dish coated with HCTA-MDI copoiymer was sterilized by u.v. irradiation and applied to the cell culture. The dish coated with deacetylated HCTA-MDI copolymer (DA-copolymer) was prepared as follows: HCTA-MDI copolymer was coated on a tissue culture plastic dish. The dish coated with HCTA-MDI copolymer was treated with 0.2M NaOMe in MeOH solution (40 ml) at room temperature for 3 h, followed by washing with MeOH, and dried in vacuo. The dish coated with regenerated cellulose was also prepared from cellulose triacetate as described above. Cell line and culture conditions A mouse fibroblast cell line (3T6 cells) 9 and a mouse

macrophage cell line (A640-BB-2 cells) 1° were used in this study. A640-BB-2 cells are clonal and immortal populations obtained from BALB/cAnN mouse bone marrow cells infected by tsA640 (temperature-sensitive gene A mutant of SV40). 3T6 cells were subcultured in a 35 × 10 mm or a 60 × 15 mm tissue culture plastic dish (Falcon Labware, Div. Becton Dickinson Oversea, Oxnard, CA, USA; Catalogue No. 3001 and 3002) of Dulbecco's modified Eagle's medium (DEM) containing 0.1% D-glucose supplemented with 7.5% heat-inactivated fetal calf serum (Hazleton, Research Products Inc. Lot No. 300509), penicillin G (500 U/ml), and streptomycin (100 #g/ml) at 37°C in a humidified incubator flushed with a 10% CO2-air mixture. A640-BB-2 cells were incubated in a bacteriological plastic dish (35 × 10 mm, Falcon Labware, Div. Becton Dickinson Oversea, Oxnard, CA, USA; Catalogue No. 1008) of DEM supplemented with 7.5% newborn bovine serum (Microbiological Associates, Walkersville, MD) in a humidified incubator flushed with a 10% CO2-air mixture at 39°C as the non-permissive temperature. Both cell colonies grown were dispersed with 0.05% trypsin (Difco Laboratories, Detroit, MI, USA) and 0.02% ethylenediamine tetra-acetate (EDTA) in phosphatebuffered saline (PBS) free of magnesium and calcium at 37°C for 10 min, and then suspended in DEM with or without fetal calf serum.

Assay for cell growth and morphology Harvested 3T6 cells and A640-BB-2 cells were reseeded at a cell density of 5.0 × 10 4 cells per 35 mm various dishes such as the bacteriological plastic dish and the dish coated with HCTA-MDI copolymer, then incubated for 4 days at 37°C and 39°C, respectively. These cells were rinsed with PBS, followed by fixing with 2% formaldehyde solution, and then stained with 5% (v/v) Giemsa solution. These specimens were examined under a light microscope BH-2 (Olympus). In the case of 3T6 cells, the dish was treated with trypsin for 15 min at 37°C, and then detached cells were stained with trypan blue dye and the number of visible cells was counted using a haemocytometer. Assay for cell attachment Cell attachment was assessed as described previously ~1'~. Briefly, harvested cells were washed with DEM. A total of 5 × 105 cells in 2.0 ml of DEM were dispersed into the dish, and incubated in a CO 2 incubator at 37°C. After incubation for various intervals, the dish containing cells was shaken on a shaking apparatus at room temperature and the medium was discarded immediately. The cells attached on the dish were fixed with 3% glutaraldehyde and stained with Giemsa solution. The percentage of the attached cells was

Int. J. Biol. Macromol., 1992, Vol. 14, October

275

Copolymers containin9 cellulose triacetate: Y. Shigemasa et al. calculated as follows: Percentage of attached cells (%) =

number ofcellsattached x 100 number ofcellsdispersed

Results and discussion

Copolymerization of hydrolysed cellulose triacetate ( HCTA) and diphenylmethane diisocyanate ( MDI) Although block copolymers prepared from several diisocyanates and cellulose triacetate oligomeric species having hydroxyl end groups were reported t-3, the

reaction conditions giving a high molecular weight of copolymer were not described in detail. Therefore, we have investigated the optimum conditions to obtain high molecular weight of H C T A - M D I copolymer. In Table 1, the effects of monomer ratio of MDI and HCTA on the copolymerization are shown. The copolymer of high molecular weight was obtained by the addition of around 1.2 mol equiv of MDI to HCTA. The mixture became a gel in 20 h at 33°C when 1.4 mol equiv of MDI to HCTA was added. Table 2 shows the effect of catalysts on the copolymerization. The copolymerization proceeded smoothly by the addition of catalyst such as triethylamine (TEA), dibutyltin dilaurate

Figure 1 Photomicrographs of attachment of mouse fibroblast cells (3T6 cells) and mouse macrophage cells (A640-BB-2 cells). The final magnification, x 100. Bacteriological plastic dish: (a) 3T6 cells after 1 day; (b) 3T6 cells after 4 days; (c) A640-BB-2 cells after 4 days. Dish coated with HCTA-MDI copolymer: (d) 3T6 cells after 1 day; (e) 3T6 cells after 4 days; (f) A640-BB-2 cells after 4 days

276

Int. J. Biol. Macromol., 1992, Vol. 14, October

Copolymers containin9 cellulose triacetate: Y. Shiyemasa et al. Table 1 Effect of monomer ratio on copolymerizationa [MDI]/[HCTA] Run

(mol/mol)

M b

Mw/M. 3.7 7.9 8.8

1

0.7

74~

2 3 4

1.0 1,2 1.4

7400 95~ ~lation

"HCTA (MW = 1700), 1 g; catalyst, triethylamine; solvent, CH2C12; temp, 33°C; time, 48 h bMolecular weight was determined by g.p.c, with pullulan as the standard

Table 2 Effect of catalyst on copolymerization~ Run

Catalyst

Time (h)

M, b

Mw/ M n

1

--

2c 3 4

-TEA DBTDL

72 72 40 37

1800 2100 7400 5500

1.6 1.7 7.9 --

addition of 1.4 mol equiv of M D I (Table 1, run 4). The gelation would be cauSed by the formation of allophanate branching between the secondary amino group of urethane linkage ( - N H C O O - ) and the isocyanate group of M D I in the presence of triethylamine. The i.r. spectra of H C T A - M D I copolymer showed an absorption band at 1520 c m - 1 which was ascribed to N H deformation of urethane linkage. The structure of the copolymer was further confirmed by 1H n.m.r, spectroscopy in D M S O - d 6. Chemical shifts at 6 = 3 . 6 - 5 . 2 p p m are attributed to the D-glucose residue of HCTA. Phenylene proton peaks appeared at 6 = 7.0-8.0 ppm. These results suggest that H C T A was linked to M D I with urethane linkage. These copolymers dissolved in various organic solvents such as CH2C12, D M F , and dimethylsulphoxide. Hence, it is thought that they have little crosslinking structure. Behaviour o f animal cells on dish Figure 1 shows the attachment of 3T6 cells to the bacteriological plastic dish and the dish coated with H C T A - M D I copolymer. When 3T6 cells were cultured on the bacteriological plastic dish for 1 or 4 days,

aHCTA ( M W = 1700), l g; temp, 33°C; [ M D I ] / [ H C T A ] , 1.0 (mol/mol); solvent, CH2C12 bMolecular weight was determined by g.p.c, with pullulan as the standard C [ M D I ] / [ H C T A ] , 1.6 (mol/mol)

(a)

100

o~

"o

10

e" U R$

I/I

Ill

:5

u

erum (-i

_= tJ

19

(b) 100

~.1.0 .Q E Z ¢U

~o

0.5

"0 eU

0

1

2 3 Time / d a y

5O

q

Figure 2 Time courses of growth of mouse fibroblast cells (3T6 cells); n = 3 . ( O ) Dish coated with HCTA-MDI copolymer; ( O ) bacteriological plastic dish

_=

u 0

( D B T D L ) , and so on. In the absence of catalyst, however, the reaction did not proceed under the reaction condition studied. In these conditions, the azeotropic distillation prior to the copolymerization was important because the water in the reaction system reacted with the isocyanate group of M D I . The gelation product was obtained by the

0

2

g

6

Time / h

Figure 3 Time courses of cell attachment to various dishes in the absence (a) and presence of fetal calf serum (b). Cell, 3T6 mouse fibroblast; n = 3. ( O ) Dish coated with HCTA-MDI copolymer; (/k) dish coated with DA-copolymer; ( O ) tissue culture plastic dish; ( • ) dish coated with regenerated cellulose

Int. J. Biol. Macromol., 1992, Vol. 14, October

277

Copolymers containing cellulose triacetate: Y. Shigemasa et al. attachment of these cells to the dish was not so good (Figure la and Figure lb). On the other hand, good attachment of 3T6 cells was observed in the dish coated with HCTA-MDI copolymer after 1 day (Fioure ld). Furthermore, 3T6 cells grew sufficiently on the dish after 4 days (Figure le). A640-BB-2 cells spread well on the dish coated with HCTA-MDI copolymer (Figure lf), though poorly on the bacteriological plastic dish

(Figure lc). In Figure 2, the time courses of growth of 3T6 cells are shown. The cell growth on the dish coated with HCTA-MDI copolymer was higher than that on the bacteriological plastic dish. These results suggest that HCTA-MDI copolymer has an effect on the growth of mouse fibroblast ceils. Next, the cell attachment to the dish coated with HCTA-MDI copolymer was compared with that of tissue culture plastic dish in the absence and presence of serum (Figure 3). In the absence of serum (Figure 3a), both the dish coated with HCTA-MDI copolymer and tissue culture plastic dish showed good cell attachment. In the presence of serum (Figure3b), however, 3T6 cells attached less to the dish coated with HCTA-MDI copolymer than to the tissue culture plastic dish. Figure 3 also shows the 3T6 cell attachment to dishes coated with deacetylated HCTA-MDI copolymer (DA-copolymer) or regenerated cellulose prepared by the deacetylation of cellulose triacetate. In the presence and absence of serum, 3T6 cells attached better to the dish coated with DA-copolymer than to the dish coated with regenerated cellulose. The structural effect of various copolymers on the cell attachment is not completely clarified at present. These results, however, suggest that urethane linkage, MDI segment, and so on would stimulate the attachment of 3T6 cells. In the present study, the dish coated with HCTA-MDI copolymer and deacetylated HCTA-MDI copolymer showed good compatibility and non-toxicity against

278

Int. J. Biol. Macromol., 1992, Vol. 14, October

animal cells such as a mouse macrophage and a mouse fibroblast. Moreover, these cells were sufficiently active on these dishes for a long period. Although the comparison of the cell attachment to these copolymers with that to polyurethanes was not examined, HCTAMDI copolymer showed almost the same cell attachment as the tissue culture plastic dish. Moreover, both HCTA-MDI copolymer and deacetylated copolymer showed higher cell attachment than regenerated cellulose. From these results, these copolymers would seem to be useful as the substratum of cell culture or biomedical materials.

Acknowledgement This work was supported in part by a grant from Toyobo Co., Ltd, which the authors gratefully acknowledge.

References 1 2 3 4 5 6 7 8 9 10 11 12

Steinmann, H. W. Polym. Prepr. 1970, 11, 285 Kim, S., Stannett, V. T. and Gilbert, R. D. J. Polym. Sci., Polym. Lett. Ed. 1973, 11, 731 Amick, R., Gilbert, R. D. and Stannett, V. Polymer 1980, 21,648 Imanishi, Y. 'Iyo Kobunshi Zairyo', Kyoritsu Shuppan, Tokyo, 1986, Chapter 3.3 Otsu, T. and Kinoshita, M. 'Kobunshi Gosei no Jikkenho', Kagaku Dojin, Kyoto, •972, Chapter 11.3, p 299 Imai, Y. 'Kagaku Sosetsu' (Ed. Nippon Kagaku Kai), No. 21, 1978, p 55 Kobayashi, A., Akaike, T., Kobayashi, K. and Sumitomo, H. Makromol. Chem. Rapid Commun. 1986, 7, 645 Sharon, N. Trends Biochem. Sci. 1984, 9, 198 Tanigawa, T., Takayama, H., Takagi, A. and Kimura, G. J. Cell. PhysioL 1983, 116, 303 Aaronson, S. A. and Todaro, J. G. J. Cell. Physiol. 1968, 72, 141 Katsumoto, T., Takayama, H. and Takagi, A. J. Electron Microsci. 1978, 27, 275 Takayama, H., Tanigawa, T., Takagi, A. and Hatada, K. Biomedical Res. 1986, 7, 11